Protein-protein interactions are fundamental to all living systems. Dynamic association and dissociation of protein complexes control most cellular functions, including cell cycle progression, signal transduction, and metabolic pathways. Complexes of transcription factors, co-repressors, and chromatin-binding proteins maintain normal cells in a quiescent state, and disruption of these protein interactions may be significant in permitting unregulated growth of cancer cells (1).
On a whole organism scale, protein-protein interactions regulate signals that affect overall homeostasis, patterns of development, normal physiology and disease in living animals. Examples include homo- and hetero-dimers of different homeobox proteins that control limb bud and craniofacial development (2), and interactions among various cell surface receptors, scaffold proteins, and transcription factors that regulate activation and trafficking of immune cells (3).
Additionally, protein interactions in signaling pathways have emerged as important therapeutic targets for cancer and other human diseases (4, 5). However, these pathways of protein interactions in specific tissues produce regional effects that cannot be investigated fully with in vitro systems and thus, there is considerable interest in imaging protein-protein interactions noninvasively in their normal physiological context within living animals with positron emission tomography (PET) (6, 7) or bioluminescence imaging (8, 9).
Regulated protein-protein interactions are fundamental to living systems, mediating many cellular functions, including cell cycle progression, signal transduction, and metabolic pathways (1, 2). In cancer, aberrant patterns of protein interactions arise from dysregulated phosphorylation of receptor tyrosine kinases (e.g., EGFR, Erb2/HER2), tumor suppressors (e.g., p53, PTEN) and targets that mediate downstream signaling in cell proliferation, survival and growth (e.g., STATs, mTOR, PI3K-Akt) (3). Thus, protein kinases and mediated protein-protein interactions comprising the kinome have emerged as important therapeutic targets in cancer and other human diseases (3-6). However, many protein interactions arise from host-cell interactions in tissue-specific pathways that cannot be investigated fully with in vitro systems and thus, there is considerable interest in imaging protein-protein interactions noninvasively in their normal physiological context within living animals with positron emission tomography (PET) (7, 8) or bioluminescence imaging (9, 10).
Current strategies for detecting protein-protein interactions include activation of transcription, repression of transcription, activation of signal transduction pathways or reconstitution of a disrupted enzymatic activity (8, 11, 12). In particular, protein fragment complementation depends on division of a monomeric enzyme into two separate components that do not spontaneously reassemble and function (13, 14). Enzyme activity occurs only upon complementation induced by the interaction of fused protein binding partners or by small molecules (drugs) that induce the interaction of fused proteins (FIG. 1). Of the available complementation strategies, feasibility studies with luciferase complementation have demonstrated the potential to observe protein-protein interactions in living animals (15, 16). However, available firefly luciferase fragments suffer from constitutive activity of the N-terminus fragment, while the blue-green emission spectrum of Renilla luciferase penetrates tissues poorly, thereby precluding general use. Furthermore, coelenterazine, the chromophoric substrate for Renilla luciferase, is transported by MDR1 P-glycoprotein (17), complicating applications of Renilla luciferase in vitro. Thus, no enzyme fragment pair yet has been found that satisfies all criteria for noninvasive analysis of protein-protein interactions and enables interrogation in cell lysates, intact cells and living animals.
Most strategies for detecting protein-protein interactions in intact cells are based on fusion of the pair of interacting molecules to defined protein elements to reconstitute a biological or biochemical function. Examples of reconstituted activities include activation of transcription, repression of transcription, activation of signal transduction pathways or reconstitution of a disrupted enzymatic activity (Toby and Golemis, 2001).
A variety of these techniques have been developed to investigate protein-protein interactions in cultured cells. The two-hybrid system is the most widely applied method to identify and characterize protein interactions. However, several features of protein fragment complementation make it attractive as an approach for in vivo imaging of protein interactions in cells, and particularly, in live animals. Below the inventors describe major features of these two methods and compare their potential utility for in vivo imaging in relation to other strategies based on our experience and published work.
Two-hybrid systems exploit the modular nature of transcription factors, many of which can be separated into discrete DNA-binding and activation domains (Fields and Song, 1989). Proteins of interest are expressed as fusions with either a DNA-binding domain (BD) or activation domain (AD), creating hybrid proteins. If the hybrid proteins bind to each other as a result of interaction between the proteins of interest, then the separate BD and AD of the transcription factor are brought together within the cell nucleus to drive expression of a reporter gene. In the absence of specific interaction between the hybrid proteins, the reporter gene is not expressed because the BD and AD do not associate independently. Two-hybrid assays can detect transient and/or unstable interactions between proteins, and the technique is reported to be independent of expression of endogenous proteins (von Mering et al., 2002).
Although the two-hybrid assay originally was developed in yeast, commercial systems (BD Biosciences Clontech) are now available for studies in bacteria and mammalian cells. The inventors and other investigators have shown that two-hybrid systems can be used to image protein interactions in living mice with positron emission tomography (PET) (Luker et al., 2002; Luker et al., 2003a; Luker et al., 2003c, b) or bioluminescence imaging (Ray et al., 2002). However, the two-hybrid method has some limitations. Some types of proteins do not lend themselves to study by the two-hybrid method. For example, because production of signal in the two-hybrid method requires nuclear localization of the hybrid proteins, membrane proteins cannot be studied in their intact state. Also, the time delay associated with both transcriptional activation of the reporter gene and degradation of the reporter protein and mRNA limits kinetic analysis of protein interactions (Rossi et al., 2000).
Protein-fragment complementation (PFC) assays depend on division of a monomeric reporter enzyme into two separate inactive components that can reconstitute function upon association. When these reporter fragments are fused to interacting proteins, the reporter is re-activated upon association of the interacting proteins. PFC strategies based on several enzymes, including β-galactosidase, dihydrofolate reductase (DHFR), β-lactamase, and luciferase have been used to monitor protein-protein interactions in mammalian cells (Rossi et al., 1997; Remy and Michnick, 1999; Remy et al., 1999; Ozawa et al., 2001; Galarneau et al., 2002; Wehrman et al., 2002).
A fundamental advantage of PFC is that the hybrid proteins directly reconstitute enzymatic activity of the reporter. In principle, therefore, protein interactions may be detected in any subcellular compartment, and assembly of protein complexes may be monitored in real time. A disadvantage of prior complementation approaches is that re-assembly of an enzyme may be susceptible to steric constraints imposed by the interacting proteins. Another potential limitation of PFC for application in living animals is that transient interactions between proteins may produce insufficient amounts of active enzyme to allow non-invasive detection. Nonetheless, because most PFC strategies are based on reconstituting active enzymes, these systems offer the potential benefits of signal amplification to enhance sensitivity for detecting interacting proteins in living animals.
The split-ubiquitin system enables signal amplification from a transcription factor-mediated reporter readout (Johnsson and Varshavsky, 1994; Stagljar et al., 1998). In one application, the interaction of two membrane proteins forces reconstitution of two halves of ubiquitin, leading to a cleavage event mediated by ubiquitin-specific proteases that release an artificial transcription factor to activate a reporter gene. As above, indirect readout of the reporter limits kinetic analysis, and the released transcription factor must translocate to the nucleus.
Several variations of recruitment systems have been developed for use in whole cells, including the Ras-recruitment system (Aronheim et al., 1997; Broder et al., 1998) and interaction traps (Eyckerman et al., 2001). Cells that co-express a test protein fused to a membrane localization signal, such as a myristoylation sequence, and a protein binding partner fused to cytoplasmic protein, such as activated Ras devoid of its membrane targeting signal, will localize mammalian Ras to the membrane only in the presence of interacting proteins. However, Ras-recruitment systems, as originally configured, cannot be applied to mammalian cells, and, while readout is not directly dependent on transcriptional activation, indirect readout by colony growth nonetheless limits kinetic analysis and interrogation of subcellular compartmentation of the interactions.
A testing variation of the recruitment approach applicable to mammalian cells is the cytokine-receptor-based interaction trap. Here, a signaling-deficient receptor provides a scaffold for recruitment of interacting fusion proteins that phosphorylate endogenous STAT3. Activated STAT complexes then drive a nuclear reporter (Eyckerman et al., 2001). This system permits detection of both modification-independent and phosphorylation-dependent interactions in intact mammalian cells, but the transcriptional readout again limits kinetic analysis.
Other approaches to detecting protein-protein interactions in live mammalian cells include fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) (Gautier et al., 2001; Boute et al., 2002). For FRET, fluorescently labeled proteins, one coupled to a donor fluorophore and the other coupled to an acceptor fluorophore, produce a characteristic shift in the emission spectra when the protein binding partners interact. A limitation of FRET is inter- and intra-molecular spatial constraints and sensitivity of detection since there is no amplification of the signal. For BRET, the donor molecule is firefly luciferase or a related bioluminescent protein, while the acceptor is green fluorescent protein or a color variant. While intermolecular spatial constraints are thought to be less restrictive with BRET, similar limitations related to sensitivity may apply. However, since the photon donor in BRET is produced by an enzymatic activity (luciferase), the potential for substrate-dependent signal amplification exists. Nonetheless, both suffer from issues related to spectral overlap that can render quantitative analysis of the two interacting fragments difficult in whole cells when expression levels are not exactly matched.
Detecting protein-protein interactions in living animals is difficult. Of the available strategies, complementation of firefly and Renilla luciferases are readily amenable to near real time applications in living animals (Ozawa et al., 2001; Paulmurugan and Gambhir, 2003), but the available fragments unfortunately suffer from considerable constitutive activity of the N-terminus fragments, thereby precluding general use of this strategy.
Thus a continuing need exits for a noninvasive analysis of protein-protein interactions. And so despite the growing body of knowledge in this area, there is still a strong and continuing need in the art to learn the identity and function of proteins engaging in protein protein interactions. In addition, because there is an unmet need to identify therapeutic compounds which modulate such activity there is also need in the art for the continued discovery in this area.
The discovery is described hereinafter in further detail with references to the aforedescribed FIGS. 1-16 in which like items are numbered the same in the aforedescribed Figures.